In recent years, a detailed understanding of the many biosynthetic pathways that can be used for the production of biofuel feedstocks and higher value bioproducts has emerged, and novel pathways for the production of specific bioenergy carriers are continuously being discovered in a variety of organisms. (Steen, E. J. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559-562 (2010); Radakovits, R., Jinkerson, R. E., Darzins, A. & Posewitz, M. C. Genetic engineering of algae for enhanced biofuel production. Eukaryotic Cell 9, 486-501 (2010); Rude, M. A. & Schirmer, A. New microbial fuels: a biotech perspective. Current Opinion in Microbiology 12, 274-281 (2009); Jang, Y.-S. et al. Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnology Advances (2011); Li, H., Cann, A. F. & Liao, J. C. Biofuels: Biomolecular engineering fundamentals and advances. Annual Review of Chemical and Biomolecular Engineering 1, 19-36 (2010)).
Further improvements in strain productivity have been hampered by the lack of a genetically tractable model system for these highly productive oleaginous algae. Currently, the algal model species are the green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, both of which have genome sequences and established transformation methods. (Merchant, S. S. et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science, 245-250 (2007); Bowler, C. et al. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature, 239-244 (2008); Siaut, M. et al. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene, 23-35 (2007); Zaslayskaia, L. A., Lippmeier, J. C., Kroth, P. G., Grossman, A. R. & Apt, K. E. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. Journal of Phycology, 379-386 (2000); Boynton, J. et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 1534-1538 (1988); Kindle, K. L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences, 1228-1232 (1990)). Genetic engineering approaches have been used to improve biofuel phenotypes in both of these organisms (Radakovits, R., Eduafo, P. M. & Posewitz, M. C. Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metabolic Engineering, 89-95 (2011); Work, V. H. et al. Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains. Eukaryotic Cell, 1251-1261 (2010); Wang, Z. T., Ullrich, N., Joo, S., Waffenschmidt, S. & Goodenough, U. Algal Lipid Bodies: Stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii. Eukaryotic Cell, 1856-1868 (2009); Li, Y. et al. Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol. Metabolic Engineering, 387-391 (2010)), unfortunately neither of these algae in their native form produce high amounts of biomass or lipids and as such, extensive genetic modifications will be needed prior to their use in biofuel applications.
Nannochloropsis is an algae that can accumulate biomass through photoautotrophy, it also stores lipids (Rodolfi, L. et al. Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnology and Bioengineering, 100-112 (2009); Converti, A., Casazza, A. A., Ortiz, E. Y., Perego, P. & Del Borghi, M. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chemical Engineering and Processing: Process Intensification, 1146-1151 (2009); Gouveia, L. & Oliveira, A. Microalgae as a raw material for biofuels production. Journal of Industrial Microbiology & Biotechnology, 269-274 (2009); Pal, D., Khozin-Goldberg, I., Cohen, Z. & Boussiba, S. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Applied Microbiology and Biotechnology, 1429-1441 (2011); Zou, N., Zhang, C., Cohen, Z. & Richmond, A. Production of cell mass and eicosapentaenoic acid (EPA) in ultrahigh cell density cultures of Nannochloropsis sp. (Eustigmatophyceae). European Journal of Phycology, 127-133 (2000)) and may be cultivated using natural sunlight in either open ponds or enclosed systems by companies such as Solix Biofuels (Fort Collins, Colo.), Seambiotic (Tel Aviv, Israel), Hairong Electric Company/Seambiotic (Penglai, China) and Proviron (Antwerp, Belgium).
What is needed is an alga that has high lipid and biomass production, whose genome sequence is know, with established protocols for genetic manipulation, and can be cultivated at commercial scale.